Mesenchymal stem cells pretreated with interferon-gamma attenuate renal fibrosis by enhancing regulatory T cell induction

Mesenchymal stem cells (MSCs) exert their anti-inflammatory and anti-fibrotic effects by secreting various humoral factors. Interferon-gamma (IFN-γ) can enhance these effects of MSCs, and enhancement of regulatory T (Treg) cell induction is thought to be an underlying mechanism. However, the extent to which Treg cell induction by MSCs pretreated with IFN-γ (IFN-γ MSCs) ameliorates renal fibrosis remains unknown. In this study, we investigated the effects of Treg cell induction by IFN-γ MSCs on renal inflammation and fibrosis using an siRNA knockdown system. Administration of IFN-γ MSCs induced Treg cells and inhibited infiltration of inflammatory cells in ischemia reperfusion injury (IRI) rats more drastically than control MSCs without IFN-γ pretreatment. In addition, administration of IFN-γ MSCs more significantly attenuated renal fibrosis compared with control MSCs. Indoleamine 2,3-dioxygenase (IDO) expression levels in conditioned medium from MSCs were enhanced by IFN-γ pretreatment. Moreover, IDO1 knockdown in IFN-γ MSCs reduced their anti-inflammatory and anti-fibrotic effects in IRI rats by reducing Treg cell induction. Our findings suggest that the increase of Treg cells induced by enhanced secretion of IDO by IFN-γ MSCs played a pivotal role in their anti-fibrotic effects. Administration of IFN-γ MSCs may potentially be a useful therapy to prevent renal fibrosis progression.

The prevalence of acute kidney injury (AKI) has been increasing in recent decades and is recognized as a worldwide health problem 1,2 .AKI is associated with subsequent development of chronic kidney disease (CKD) 3 .Therefore, mechanisms underlying AKI-to-CKD progression have been extensively investigated over the past decade.The main pathological feature of AKI-to-CKD progression is interstitial fibrosis, which can result from one or more pathological mechanisms, such as hypoxia 4 , endothelial dysfunction, microvascular rarefaction 5 , inflammation 6 , transforming growth factor (TGF)-β1 production 7 , and epithelial-mesenchymal transition (EMT) 8 .Notably, among these mechanisms, inflammation plays a major role in fibrosis progression 9 .
Numerous studies have reported that sustained pro-inflammatory stimuli activate fibroblasts, resulting in fibrosis 10,11 .In recent years, regulatory T (Treg) cells have received much attention for their role as an inhibitory system against this progressive inflammation 12,13 .Treg cells can reportedly suppress excessive immune responses by various mechanisms, including direct cell-cell contact 14 , depletion of interleukin (IL)-2 15,16 , and release of soluble inhibitory factors like IL-10 17,18 .In addition, Treg cells exert protective effects against renal injury 19 .Therefore, increasing Treg cell induction within the injury site is potentially a novel therapeutic strategy for renal fibrosis.
Mesenchymal stem cells (MSCs) are multipotent adult stem cells 20 isolated from various tissues, such as bone marrow, adipose tissue, and umbilical cord 21 .We elucidated that the anti-fibrotic effects of xenogeneic and allogeneic MSCs are almost equal 22 .Moreover, many studies have revealed that xeno therapy using human MSCs is effective for mouse models of renal injury 23,24 .MSCs contribute to tissue regeneration by secreting various soluble factors 25,26 .MSCs are known to favor the generation of Treg cells, producing anti-inflammatory effects 27 .
In this study, we investigated the effects of Treg cell induction by IFN-γ MSCs on renal inflammation and fibrosis in a unilateral renal ischemia reperfusion injury (IRI) rat model with contralateral nephrectomy using a siRNA knockdown system.

MSCs pretreated with IFN-γ inhibited inflammatory cell infiltration by inducing regulatory T cells
We first investigated whether IFN-γ MSCs had a stronger immunosuppressive effect than untreated MSCs in an experimental IRI model.After the IRI procedure, 5 × 10 5 cells untreated MSCs (control MSCs), IFN-γ MSCs, or PBS was injected into rats through the abdominal aorta.At 7 days after IRI, immunohistochemical staining showed that numbers of cells positive for FOXP3, a Treg marker, were not drastically changed in IRI rats injected with PBS compared with the findings in the control group.Meanwhile, numbers of FOXP3-positive cells were increased in IRI rats injected with control MSCs and further upregulated in IRI rats injected with IFN-γ MSCs (Fig. 1a,b).In contrast, numbers of cells positive for CD3 (a pan T cell marker) and CD68 (a macrophage marker) were increased in the PBS group.These observations of increased expression were suppressed in the control MSCs group, and further suppressed in the IFN-γ MSCs group (Fig. 1a,b).We also performed double immunostaining for FOXP3 and CD3 to identify Treg cells.Positive cells for both FOXP3 and CD3 were increased in the MSCs group, and a further increase was observed in the IFN-γ MSCs group (Fig. 1a,b).

MSCs pretreated with IFN-γ increased vascular endothelial growth factor in IRI rats
Upregulation of vascular endothelial growth factor (VEGF) in IRI rats is critical to prevent AKI progression.Therefore, we investigated the effects of IFN-γ MSCs on VEGFA expression in IRI rats at 7 days post-injection.The protein level of VEGFA was upregulated in the control MSCs group and further upregulation was observed in the IFN-γ MSCs group (Fig. 1c, Additional File 1A: Fig. S1a).

MSCs pretreated with IFN-γ suppressed IRI-induced tubulointerstitial fibrosis and extracellular matrix accumulation
Next, we investigated the effect of IFN-γ MSCs on tubulointerstitial fibrosis in IRI rats at 21 days post-injection.We found that protein levels of α-smooth muscle actin (α-SMA) and TGF-β1 were remarkably increased in the PBS group, suppressed in the control MSCs group, and further suppressed in the IFN-γ MSCs group (Fig. 2a,b, Additional File 1A: Fig. S1b,c).Masson's trichrome staining revealed that the extent (area) of interstitial fibrosis was more significantly suppressed in the IFN-γ MSCs group compared with that in the control MSCs group.Similarly, α-SMA-and collagen type I-positive areas were more significantly reduced in the IFN-γ MSCs group compared with those in the control MSCs group (Fig. 2c,d).

IFN-γ pretreatment increased indoleamine 2,3-dioxygenase expression in conditioned medium from MSCs
Previous studies demonstrated that IDO expression plays a direct role in inducing the conversion of naïve CD4 T cells into Treg cells 30,31 .Therefore, we examined IDO expression levels in MSCs and CM collected from MSCs.We found that mRNA and protein levels of IDO were more significantly upregulated in IFN-γ MSCs compared with those in control MSCs (Fig. 3a,b, Additional File 1B: Fig. S1d).Additionally, ELISA results show that the concentration of IDO in IFN-γ MSCs-CM was higher than in control MSCs-CM (Fig. 3c).

Conditioned medium from IFN-γ MSCs enhanced differentiation of naïve CD4 T cells into FOXP3-positive regulatory T cells
To investigate whether IFN-γ MSCs mediated the induction of FOXP3-positive Treg cells, we isolated naïve CD4 T cells from PBMCs and cultured them with control MSCs-CM or IFN-γ MSCs-CM.IFN-γ MSCs-CM more significantly increased FOXP3 protein levels compared with control MSCs-CM (Fig. 3d, Additional File 1B: Fig. S1e).

IDO1 knockdown in IFN-γ MSCs weakened their anti-fibrotic effects in IRI rats
Finally, we investigated the extent to which Treg cell induction by IFN-γ MSCs ameliorated renal fibrosis.Western blotting revealed that protein levels of α-SMA and TGF-β1 were increased in the PBS group and significantly ameliorated by injection of NC siRNA/IFN-γ MSCs, while these reductions were completely abrogated by injection of IDO1 siRNA/IFN-γ MSCs (Fig. 5a,b, Additional File 1C: Fig. S1f,g).Similarly, immunostaining showed that α-SMA-and collagen type I-positive areas were expanded in the PBS group and markedly ameliorated by injection of NC siRNA/IFN-γ MSCs, whereas these improvements were abolished by injection of IDO1 siRNA/ IFN-γ MSCs (Fig. 5c,d).Advances in MSC biology have confirmed that sufficient inflammatory signals are required to activate the immunosuppressive properties of MSCs 32,33 .IFN-γ, a cytokine expressed in activated lymphocytes, reportedly modifies the immunomodulatory function of MSCs 34,35 .As a mechanism by which MSCs exert an anti-inflammatory effect, we previously showed that IFN-γ MSCs increase prostaglandin E2 (PGE2) secretion to induce immunosuppressive M2 macrophage polarization, leading to enhanced anti-inflammatory and anti-fibrotic effects 36 .The present study demonstrates that IFN-γ MSCs enhanced IDO secretion, thereby inhibiting infiltration of inflammatory cells by inducing Treg cells, leading to pronounced anti-fibrotic effects.Taken together, our findings indicate that IFN-γ MSCs enhanced the suppression of inflammatory cell infiltration through two pathways: induction of M2 macrophages by increasing PGE2 secretion and induction of Treg cells by upregulating IDO secretion.MSCs exert immunosuppressive and therapeutic effects by a Treg cell-induced mechanism in various diseases models, such as transplantation 37,38 , autoimmune disorders 39,40 , and systemic inflammatory diseases 41,42 .MSCs remain dormant under normal conditions and exert anti-inflammatory effects when activated by cytokines www.nature.com/scientificreports/released from immune cells in damaged tissues.This activation may require several days and some MSCs may be unable to change to the active form 36 , so normal MSCs may not exert sufficient therapeutic effects.In fact, our

Figure 1 .
Figure 1.Anti-inflammatory effects of mesenchymal stem cells (MSCs) by regulatory T (Treg) cells induction in the kidney of ischemia reperfusion injury (IRI) rats.MSCs pretreated with IFN-γ (IFN-γ MSCs) or MSCs untreated were injected immediately after IRI induction.Infiltration of T cells and macrophages into injured kidney of MSC-injected IRI rats was assessed at day 7 post-IRI by immunostaining.(a) Representative image of immunohistochemical staining of FOXP3, CD3, and CD68 and double immunostaining of FOXP3 (brown) and CD3 (blue) in kidney sections (scale bar = 100 µm).(b) Quantification of FOXP3-, CD3-, CD68-positive cells and double-positive (FOXP3 and CD3) cells (n = 5 in each group).(c) Western blot analysis of VEGFA in the kidney cortex of IRI rats.Graphs show densitometric analyses of VEGFA expression levels normalized to the GAPDH expression level (n = 5 in each group).Full-length blots are presented in Supplementary Fig. 1a.Data indicate mean ± S.D. # P < 0.01, *P < 0.05 (one-way ANOVA followed by Bonferroni's post-hoc test).

Figure 2 . 4 Discussion
Figure 2. Anti-fibrotic effects of IFN-γ MSCs in the kidney of IRI rats.Protein levels of fibrosis markers were evaluated at day 21 post-IRI by western blot (a, b), Masson's trichrome staining and immunohistochemical (c) analyses.(a, b) Western blot analysis of α-SMA and TGF-β1 in the kidney cortex of IRI rats.Graphs show densitometric analyses of α-SMA and TGF-β1 expression levels normalized to the GAPDH expression level (n = 5 in each group).Full-length blots are presented in Supplementary Fig. 1b, 1c.(c) Representative images of Masson's trichrome staining and immunohistochemical staining of α-SMA and collagen type I in kidney sections (scale bar = 100 µm).(d) Quantification of interstitial fibrosis area and α-SMA-and collagen type I-positive areas as percentages of the total area (n = 5 in each group).Data indicate mean ± S.D. # P < 0.01, *P < 0.05 (one-way ANOVA followed by Bonferroni's post-hoc test).

Figure 3 .
Figure 3. Effects of conditioned medium obtained from IFN-γ MSCs on differentiation of naïve CD4 T cells into regulatory T cells.mRNA and protein levels of indoleamine 2,3-dioxygenase (IDO) were evaluated by quantitative real-time polymerase chain reaction (a) and western blot (b) analyses.(a) Expression levels of IDO1 mRNA in HK-2 cells, MSCs, and IFN-γ MSCs (n = 5 in each group).(b) Western blot analysis of IDO in HK-2 cells, MSCs, and IFN-γ MSCs.Graph shows densitometric analysis of IDO expression levels normalized to the GAPDH expression level (n = 5 in each group).Full-length blots are presented in Supplementary Fig. 1d.(c) Concentration of IDO in conditioned medium from MSCs was evaluated by an ELISA analysis at 48 h after IFN-γ stimulation.DMEM containing 0.1% FBS was used as a negative control.Graph shows IDO concentrations in DMEM containing 0.1% FBS or conditioned medium from MSCs or IFN-γ MSCs (n = 5 in each group).(d) Differentiation of naïve CD4 T cells into regulatory T cells were evaluated by FOXP3 and CD4 protein levels.Naïve CD4 T cells were isolated from human peripheral blood mononuclear cells and were incubated with DMEM containing 0.1% FBS or conditioned medium obtained from MSCs or IFN-γ MSCs.Five days later, the cells were harvested and subjected to western blot analysis of FOXP3 and CD4.Graph shows densitometric analysis of FOXP3/CD4 expression levels normalized to the GAPDH expression level (n = 5 in each group).Full-length blots are presented in Supplementary Fig. 1e.Data indicate mean ± S.D. # P < 0.01 (oneway ANOVA followed by Bonferroni's post-hoc test).

Figure 5 .
Figure 5. Inhibitory effects of IDO1 siRNA on anti-fibrotic effects of IFN-γ MSCs in IRI rats.After MSCs transfected with NC siRNA or IDO1 siRNA were pretreated with IFN-γ, the cells were injected into rats immediately after IRI induction.Protein levels of fibrosis markers were evaluated at day 21 post-IRI by western blot (a, b), Masson's trichrome staining and immunohistochemical (c) analyses.(a, b) Western blot analysis of α-SMA and TGF-β1 in the kidney cortex of IRI rats.Graphs show densitometric analyses of α-SMA and TGF-β1 expression levels normalized to the GAPDH expression level (n = 5 in each group).Full-length blots are presented in Supplementary Fig. 1f, 1g.(c) Representative images of Masson's trichrome staining and immunohistochemical staining of α-SMA and collagen type I of kidney sections (scale bar = 100 µm).(d) Quantification of interstitial fibrosis area and α-SMA-and collagen type I-positive areas as percentages of the total area (n = 5 in each group).Data indicate mean ± S.D. # P < 0.01, *P < 0.05 (one-way ANOVA followed by Bonferroni's post-hoc test).